Hematopoietic stem cells (HSCs) are pluripotent cells that give rise to all lineages of mature blood cells. HSCs are ideal targets for vector-mediated gene therapy because of their ability for self-renewal and the wide distribution of their progeny. Gene transfer into HSCs has tremendous potential as a means for treating a variety of hematologic and immune disorders.
Retroviral vector systems such as oncoretroviral- and lentiviral-based systems are among the most widely used vector systems for gene therapy. The advantages of retroviral vectors include high efficiency of gene delivery, integration into the host genome, and high levels of gene expression. Vectors derived from murine leukemia virus (MLV), a prototypical oncoretrovirus, have been widely used to deliver genes into HSCs in human gene therapy trials. However, MLV integrates randomly into the host genome, which can lead to gene disruption or unanticipated gene activation through the enhancer or the promoter element in the vector (Li 2002). In a recent gene therapy trial involving the treatment of patients with severe combined immunodeficiency syndrome (SCID), vector integration either near or in the LMO-2 gene resulted in the activation of LMO-2 expression (Hacein-Bey-Abina 2003). This most likely led to leukemia development in two of the nine treated patients. Another drawback to the use of MLV-based vectors is their inability to infect and integrate into non-dividing cells (Miller 1990). This is an issue because HSCs spend the majority of their time in a quiescent state.
Lentiviruses such as human immunodeficiency virus (HIV) differ from oncoretroviruses in that their replicative cycle does not require cell division. This means that the HIV virus can integrate into the genome of non-dividing cells, which partially circumvents the problem of low transduction efficiency in quiescent cells. Upon entry into a host cell, HIV reverse transcriptase generates DNA copies of viral RNA. This DNA is bound to a variety of proteins in a complex called the preintegration complex (PIC). Proteins found in the PIC include nucleocapsid (NCp7), matrix antigen (MA), p6, integrase (IN), Tat and viral protein R (Vpr). Three of these proteins, Ma, In, and Vpr, contribute to the ability of HIV to transduce non-dividing cells, a process that depends on transport of the PIC into the host cell nucleus. Vpr plays a key regulatory role in this nuclear transport by binding to karyopherin α (Popov 1998a), which belongs to a family of cellular proteins involved in active nuclear import (Gallay 1997; Popov 1998b).
HIV vectors have been used to transduce HSCs (Sutton 1998; Uchida 1998; Case 1999; Evans 1999; Miyoshi 1999), hepatocytes (Kafri 1997), neuronal cells (Naldini 1996), and skeletal muscle cells (Kafri 1997). However, transplantation experiments performed in large animals do not support the notion that HIV vectors can transduce hematopoietic repopulating cells more efficiently than MLV vectors (An 2000; Horn 2002a; Horn 2002b). In order to achieve maximum transduction efficiency, a high multiplicity of infection (MOI) is required (Haas 2000; Salmon 2000). However, the use of a high MOI frequently leads to multi-copy vector insertion into host chromosomes (Woods 2003), which increases the risk of cancer due to random vector integration. Vector integration near an oncogene has been linked to an increase in leukemia in SCID patients receiving gene therapy (Hacein-Bey-Abina 2003). In addition, recent studies have shown preferential integration by retrovirus and HIV near or within active genes (Schroder 2002; Wu 2003). These findings underscore the importance of introducing only a limited number of vector copies into the host genome. This can be accomplished by using a low MOI, but this approach reduces transduction efficiency. If HIV vector-mediated gene therapy is going to be successful, it is important to develop approaches for site-specific gene insertion into the host genome.
For targeted integration to avoid insertion mutagenesis, the process of gene replacement by homologous recombination is a very useful but typically inefficient technique (Capecchi 1989). Using this technique in mammalian cells, gene insertion typically only occurs in about 1 out of every 106 cells treated (Capecchi 1989; Koller 1992). For such an event to occur in HSCs, it is almost a prerequisite that a viral vector such as the HIV vector be used, based on its high efficiency of gene transfer into cells. To make gene targeting practical, however, the low frequency of homologous recombination needs to be improved significantly. The use of site-specific recombinases such as Cre has been shown to significantly enhance the efficiency of gene targeting in a mammalian cell environment (Sauer 1993). Cre, a 38-kDa recombinase from bacteriophage P1, utilizes its endonuclease activity to catalyze recombination between two identical IoxP sites. The enzyme requires no accessory proteins or cofactors and functions efficiently in vitro and under a wide variety of cellular conditions (Abremski 1983; Sternberg 1981a; Sternberg 1981b; Sternberg 1981c). The recombination site recognized by Cre is a 34-base pair (bp) double-stranded DNA sequence known as IoxP. Each IoxP site consists of two 13-bp inverted repeats separated by an 8-bp asymmetrical core region. Cre binds to the inverted repeats and cleaves the DNA in the core region to facilitate DNA strand exchange reactions (Abremski 1983; Sternberg 1981a; Sternberg 1981b; Sternberg 1981c). High-level Cre expression in mammalian cells has been shown to mediate site-specific gene insertion at relatively high frequency (Vanin 1997).